Accurate DNA replication is of fundamental importance to all life ensuring the maintenance and transmission of the genome and limiting tumorigenesis in higher organisms. High-fidelity DNA polymerases perform an astonishing feat of molecular recognition, incorporating the correct nucleotide triphosphate (dNTP) substrate molecules as specified by the template base with minimal error rates. For example, even without exonucleolytic proofreading, the replicative DNA polymerase III from E. coli on average only makes one error in ˜105 base pairs (Schaaper JBC 1993).
As energetic differences between correctly and mispaired nucleotides per se are much too small to give rise to a 105 fold discrimination, the structure of the polymerase active site in high-fidelity polymerases has evolved to enhance those differences. Recent structural studies of the A-family (Pol I-like) DNA polymerases from Thermus aquaticus (Taq) (Li 98), phage T7 (Ellenberger) and B. stearothermophilus (Bst) (Beese) in particular have revealed how conformational changes during the catalytic cycle may exclude non-cognate base-pairing geometries because of steric clashes within the closed active site. As a result of these tight steric constraints, not only are mismatched nucleotides excluded but catalysis becomes exquisitely sensitive to even slight distortions in the primer-template duplex. This precludes or greatly diminishes the replication of modified or damaged DNA templates, the incorporation of modified or unnatural deoxinucleotide triphosphates (dNTP) and the extension of mismatched or unnatural 3′ termini.
While desirable in nature, such stringent substrate discrimination is limiting for many applications in biotechnology. Specifically, it restricts the use of unnatural or modified nucleotide bases and the applications they enable. It also precludes the efficient PCR amplification of damaged DNA templates.
Some other naturally occurring polymerases are less stringent with regard to their substrate specificity. For example, viral reverse transcriptases like HIV-1 reverse transcriptase or AMV reverse transcriptase and polymerases capable of translesion synthesis such as polY-family polymerases, pol X (Vaisman et al, 2001, JBC) or pol X (Washington (2002), PNAS; or the unusual polB-family polymerase pol X (Johnson, Nature), all extend 3′ mismatches with elevated efficiency compared to high fidelity polymerases. The disadvantage of the use of translesion synthesis polymerases for biotechnological uses is that they depend on cellular processivity factors for their activity, such as PCNA. Moreover such polymerases are not stable at the temperatures at which certain biotechnological techniques are performed, such as PCR. Furthermore most Translesion synthesis polymerases have a much reduced fidelity, which would severely compromise their utility for cloning.
Using another approach, the availability of high-resolution structures has guided efforts to rationally alter the substrate specificity of high fidelity DNA polymerases by site-directed mutagenesis e.g. to increase acceptance of dideoxi- (ddNTPs) (Li 99) or ribonucleotides (rNTPs) (Astatke 98). In vivo complementation followed by screening has also yielded polymerase variants with increased rNTP incorporation and limited bypass of template lesions (Patel 01). Recently, two different in vitro strategies for selection of polymerase activity have been described (Jestin 00, Ghadessy 01, Xia 02). One is based on the proximal attachent of polymerase and template-primer duplex on the same phage particle and has allowed the isolation mutants of Taq polymerase, which incorporate rNTPs and dNTPs with comparable efficiency (Xia 02). However, such methods are complex, prone to error and are laborious.
Recently, the technique of compartmentalized self-replication (CSR) (Ghadessy 01), which is based on the self-replication of polymerase genes by the encoded polymerases within discrete, non-communicating compartments has allowed the selection of mutants of Taq polymerase with increased thermostability and/or resistance to the potent inhibitor heparin (Ghadessy et al 01).
However, there still remains a need in the art for an efficient and simple method for relaxing the substrate specificity of high fidelity DNA polymerases whilst maintaining high catalytic turnover and processivity of DNA fragments up to several tens of kb. Such polymerases will be of particular use in applications such as PCR amplification and sequencing of damaged DNA templates, for the incorporation of unnatural base analogues into DNA (such as is required for sequencing or array labelling) and as a starting point for the creation of novel polymerase activities using compartmentalised self replication or other methods.